Aging is accompanied by a general decline of physiological function and significant increases in the incidence of cancer and other degenerative diseases. It has been hypothesized that alterations in apoptosis may contribute to these age-associated changes. Changes in the extent of apoptosis as a function of age have been observed in a number of tissues or cell types. However, the implication of these changes in apoptosis during aging and how the aging process itself may modify the regulatory machinery of apoptosis remains an enigma. Further confounding such studies is the fact that the contribution of different tissues to aging is not equal. Currently, the most popular explanation for how aging occurs at the biochemical/molecular level is the oxidative stress theory of aging. Oxidative damage to cellular components (and mitochondria, in particular) by reactive oxygen species (ROS) is proposed to alter the structure and function of a variety of macromolecules, e.g., DNA, lipid, and proteins. These alterations in turn can lead to reduced physiological function and eventually aging and increased pathology. Oxidative stress is also a potent inducer of mitochondrial apoptosis suggesting that oxidative stress over the lifespan of an organism may elicit its effects on aging via regulation of mitochondrial-dependent apoptosis. We have recently defined the age-dependent changes in apoptosis that occur in a number of organs during normal aging (Zhang et al., 2002, Reprint 1). Amongst our findings is that normal aging is accompanied by up regulation of the activity of the intrinsic pathway of apoptosis in regenerative but not differentiated tissues. We have generated exciting new preliminary data that implicates a specific caspase, caspase-2 as a potential mediator of mitochondrial oxidative stress in an age-dependent fashion. These data demonstrate that loss of caspase-2: a) increases resistance of cells specifically to mitochondrial oxidant stressors;b) results in a mouse which displays a premature aging phenotype (e.g. lower bone mineral density, lower fat content, decreased body weight and shorter lifespan), and c) extends the lifespan of the MnSOD-/- mouse. Collectively our findings have led us to propose the following hypotheses: 1) Caspase-2 modulates mitochondrial oxidative stress-induced apoptosis in hepatocytes and neurons and such apoptosis affects aging in a tissue specific fashion. 2) Loss of oxidative stress induced caspase-2 mediated apoptosis results in an accelerated age-related accumulation of oxidative damage, leading to a shortening of organismal lifespan. To test these hypotheses we will undertake the following specific aims: 1) Determine the age-dependent sensitivity of hepatocytes, cortical neurons, mesencephalic cells and fibroblasts obtained from caspase-2-/-, caspase-2 liver specific (caspase-2L-/-) caspase-2 brain specific (caspase-2B-/-) and WT mice to various oxidant stressors, both in vitro and in vivo. 2) Measure the survival of the caspase-2-/-, caspase-2L-/-, caspase-2B-/, MnSOD-/-, MnSOD -/-/caspase-2-/- and MnSOD -/-/caspase-2+/-'mice compared to WT mice. 3) Determine if caspase-2-/-, caspase-2L-/- and caspase-2B-/- mice are more or less resistant to oxidative stress, and whether oxidative stress and/or age result in the development of organ specific pathology. These studies are both significant and innovative, as they address mechanisms of a fundamental biological process, apoptosis, involved in a number of normal and pathological activities of organisms and will provide important information about the relationship between oxidative stress, apoptosis and aging in a tissue specific fashion.